Title:  Reparatives for ultraviolet radiation skin damage

United States Patent:  6,337,320

Assignee:  Thione International, Inc. (Altanta, GA)

Filed:  October 11, 1996

A composition of L-glutathione (reduced) and selenium and an epidermal growth factor in a topical carrier and method of using the composition to reduce and repair ultraviolet radiation-induced skin damage, both acute injury (sunburn) and chronic damage (photoaging and cutaneous malignancies).

DETAILED DESCRIPTION OF THE INVENTION

The present invention deals with reduced L-glutathione (GSH), in combination with selenium and thiol compounds used topically to act as free radical scavengers reducing ultraviolet radiation-induced skin changes. It is proposed that the described active ingredients be employed in topical compositions. Topical carriers are employed which should be both non-irritating to the skin and which are suitable for delivering the active components to the skin. Further, suitable topical carriers should be those which do not inhibit the antioxidant activity of the active ingredients thus reducing the efficiency of the composition for protecting the skin from the effects of acute and chronic ultraviolet radiation. Further, such carriers must be of sufficiently high purity and sufficiently low toxicity to render them suitable for chronic topical administration to the skin and be free of bacterial contaminants.

Certain antioxidants, particularly the endogenous L-glutathione, superoxide dismutase and acetyl L carnitine, as well as the element selenium, a co-factor for the enzyme glutathione peroxidase, and thiol compounds such as L-cysteine, can be employed in suitable carriers such as lotions, solutions, creams, ointments, balms, sprays, aerosols, gels or foundation compositions to protect and to treat the overlying skin surface as a result of the putative acute and chronic UV radiation etiologic factors in specifically dealing with the effects of the various free radicals on biomolecules, lipids, and cell membranes. Moreover, specific cellular growth factors, such as epithelial and fibroblast growth factors in appropriate concentrations and delivery vehicles, are incorporated in the preventive and reparative preparations of this invention for aiding the repair of UV radiation damage of skin and healing of the superficial wounds as occurs in sunburns and in the chronic UV radiation injury known as photoaging of the skin.

Without being bound to any particular theory, it is noted that reduced glutathione is employed in protecting cells and aerobic organisms against oxidative stress by itself being oxidized. Thus, L-glutathione must act in combination with other enzyme systems in order to be reduced so that it may renew its role as a free radical scavenger. GSH functions also coordinately with the enzyme glutathione peroxidase to break down hydrogen peroxide and lipid hydroperoxides. Glutathione peroxidase in the body requires selenium as a cofactor to exert its biologic antioxidant function. Selenium compounds have been shown to scavenge oxygen-centered radicals in vivo with reduced glutathione through glutathione peroxidase. It is believed that selenium-GSH peroxidase catalyzes toxic hydrogen peroxide in the presence of reduced glutathione. This reaction reduces glutathione to oxidized glutathione (GSSG). In turn, the GSSG is reduced back to GSH by the enzyme GSH reductase thereby maintaining abundant cellular GSH to scavenge free radicals anew. GSH reductase is provided in these preparations through thiol rich yeast extracts.

It is further contemplated that the present composition, as a preferred embodiment, includes acetyl L carnitine. This latter component further participates in protecting cells against lipid peroxidation by locally increasing the amount of antioxidizing agents of GSH and ubiquinol. L-carnitine, also known as gamma trimethylamino-beta hydroxy butyrate or Vitamin Bt, occurs naturally in the body. It is a normal endogenous intermediary metabolite which has been identified in all mammalian cells and in blood and urine. It has the function of transporting fatty acids and other acidulated compounds across inner mitochondrial membranes and of maintaining the acyl CoA/free CoA ratio between the mitochondria and the cytosol of the cells. Acetyl L carnitine is the acetyl derivative of l-carnitine and is also a naturally occurring substance in the body as it provides a transport mechanism for the acetyl groups created by the beta oxidation of fatty acids while concomitantly regenerating acetyl co-enzymes in the cytosol of the cell.

Of interest herein, acetyl L carnitine has been shown to have a scavenging effect on the free superoxide anion. This antioxidant activity coupled by acetyl L carnitine's effect of inducing an increase in reduced glutathione and reduced ubiquinone levels provides a stabilizing effect on membranes by decreasing membrane lipid peroxidation.

The skin is a highly vascular organ, extracellularly very rich in polyunsaturated fatty acids. The skin exposed to ultraviolet rays with its exposure to atmospheric oxygen is most prone to the process of lipid peroxidation and thus skin may be readily damaged acutely and/or chronically by this radiation, both UVA and UVB. Thus, reduced glutathione and acetyl L carnitine in a topical preparation will act somewhat synergistically; the former as a reparative antioxidant which itself becomes oxidized and better able to be regenerated locally in its reduced form by the metabolic functions of acetyl L carnitine and by acetyl L carnitine's ability to enhance mitochondrial energy production. This is accomplished by the latter's actions on lipid metabolism and by the resulting increase in cytochrome oxidase, the final enzyme in the cellular respiratory chain.

Further, glutathione and selenium act synergistically in vivo as they are both constituents of the same enzymatic system. GSH serves as a specific donor substrate while selenium, provided from alimentary sources or locally from topically applied preparations of selenoamino acids, selenium yeast extracts or selenoamino acid chelates, provides the prosthetic group of GSH peroxidase, during its synthesis. The glutathione and selenium antioxidant functions are intrinsically related since by keeping a peroxidase in action, the GSH and selenium, contribute to the removal of the dismutation product of free oxygen radicals, namely, hydrogen peroxide. In a broad sense, GSH and selenium modulate free radical chains initiated or sustained by hydro peroxides.

Selenium is used in the present invention for its role as an antioxidant as well as its anticarcinogenic and antimutagenic properties. Selenium is an essential trace element, and a cofactor and constituent of the enzyme glutathione peroxidase. Selenium preparations as a sulfide have been used as topical antiseborrheic detergents (Selsun.RTM.) and in veterinary medicine topically for eczemas and dermatomycoses.

Selenomethionine decomposes lipid peroxides and inhibits in vivo lipid peroxidation in tissues of vitamin E deficient chicks. Other selenoproteins also show a high degree of inhibition of lipid peroxidation in hepatic tissues of various species, thus concluding that in vivo selenium exhibits antioxidant behavior.

Selenium has also been shown to affect the immune system. Selenium supplementation as 70% selenomethione in patients with psoriasis with normal pretreatment selenium blood levels showed an increase in blood levels of 40% post treatment, although skin levels of selenium dependent glutathione peroxidase were unchanged in both normal and psoriatic skin. A statistically significant increase in the number of CD4+T-cells was noted in the reticular dermis of the psoriatic lesions.

In other studies in human subjects, optical selenomethionine was investigated for its ability to reduce the degree of acute damage to the skin by sunburn as induced experimentally by ultraviolet irradiation. Eight women were treated for two weeks with a lotion vehicle and then with three concentrations of selenomethionine (0.002%, 0.02% and 0.05%). The researchers found that topical selenomethione was effective in protecting against acute UV damage to the skin, as measured by the minimal erythema dose, using a multiport solar ultraviolet simulator. Plasma levels of selenium in these volunteers remained unchanged, suggesting the protective effect of the selenomethionine was locally at the skin.

The effects demonstrated by topical selenomethinine in human volunteers (Burke et al.) on measurement of minimal erythema dose, suggests that the protection to ultraviolet irradiation by this compound is not simply a sunscreen effect. The selenomethionine is absorbed percutaneously and acts locally as a free radical scavenger, after absorption from the outer skin layers, acting most likely as the co-factor for the enzyme glutathione peroxidase. Thus, even if the person perspires or goes swimming, selenomethionine continues to afford its protective effect as a local antioxidant and not as a sun blocker, which needs to be reapplied to the skin to render its value as a protectant or absorber of UV irradiation. No selenium toxicity is possible at the doses used in these topical compositions (0.001 to 0.05% with an average dose at 0.025%) for studies have indicated selenium toxicities occur in excess of oral 4000 micrograms per day for prolonged periods. Like all sunscreen-sun blocking preparations, the compositions disclosed herein require that the active, synergistic complex, which includes selenoamino acid or selenium yeast extract and reduced glutathione be applied topically by the individual about 30 minutes prior to the expected exposure to solar radiation. Moreover, persons expected to be exposed to solar radiation need to heed the other well known sun safety measures, including coverups with clothing, hats and sunglasses.

Compositions of reduced glutathione in the present invention comprise from about 0.001%, preferably from about 0.1% to 15%, more preferably from about 1% to 5% by weight.

The lower limit of concentration for selenomethionine is selected to achieve a composition in which its amount in the topical preparation provides a therapeutic concentration of the selenoamino acid, no lower than 0.001%. The concentrations to be employed are between 0.001% and 5%, but preferably from 0.01 to 1.0% but most preferably from 0.015 to 0.05% by weight.

"Cell growth stimulating compounds or factors" have been described as natural or exogenous compounds which have a stimulating effect on the elaboration and growth of specific cell lines. These include anabolic growth hormones, as human growth hormone and thyroid stimulating hormone, or on specific cell lines as granulocytes, platelets or erythrocytes. Specifically, in regard to promoting epidermal growth, such as in skin tissue repair or wound healing, various factors have been identified as growth factors, including epithelial (epidermal) growth factor (EGF), fibroblast growth factor (FGF), tissue respiratory factor (TRF), transforming growth factor (TGF) and insulin-like growth factor (IGF).

In the present formulations using antioxidants and anti-inflammatory compounds, one or more cell growth stimulating compounds in suitable amounts effective for stimulating the growth of cells which encompass or surround and are injured or are responsible for healing wounds may be incorporated in the preparation of the present creams, balms, lotions, solutions or gels; patches, sprays or other cosmetic and foundation compositions. Skin cellular reparative functions of dermatologic injuries or lesions (sunburn, gamma radiation and laser burns, chemosurgery, dermatoses, etc.) are included in the list of therapies as examples.

Also useful herein is a component known as tissue respiratory factor (TRF). TRF is a live yeast cell derivative which has been used in over the counter pharmaceutical preparations since the 1940's and more recently as an ingredient in cosmetics. It is commercially available (Brooks Industries Biodynes-TRF.TM., South Plainfield, N.J.) and purported to be a powerful internal moisturizer which refreshes dry and infirm skin. TRF was first used as an anti-hemorrhoidal product (Preparation H^o, Whitehall Laboratories). TRF is composed of low molecular weight glycosidic/peptide fractions, with a ratio of 1:3. The residual glycopeptide linkages are through the amino acid asparagine residues. Because TRF is prepared from live yeast cells derivatives, additional trace quantities of coenzymes, vitamins, amino acids and minerals, characteristic of yeast, are available in these factors, which enhance the therapeutic capabilities of TRF in these pharmaceutic/cosmetic preparations.

TRF has a maximum absorbance of 13.0-20.0; ultraviolet spectrophotometer of a 1% TRF filtered solution reads at 256-258 NM. It is available as a water soluble material for gels, emulsions, lotions and creams. TRF has been shown to promote wound healing through its ability to increase fibroblast synthesis of collagen and elastin fibers resulting in smoothing of the skin. TRF's moisturizing effect is accomplished by increasing uptake of moisture by nascent protein and increasing oxygen utilization in the skin. TRF has been used in the treatment of sunburned skin and has been preferred for decreasing pain and discomfort of sunburn damaged skin when compared to a topical post-sun product containing the local anesthetic benzocaine. Thus, TRF, as other growth factors, may be used in combination to these proposed antioxidant preparations as a preventive and prophylactic agent to photodamaged, burned, irradiated or inflamed skin of diverse etiologies.

A further optional expedient is the use of epidermal growth factor (EGF). Epidermal growth factor is an endogenous substance for the development and maintenance of the epidermis and dermis. EGF is a protein that catalyzes the cutaneous healing process by promoting epidermal and epithelial cells to divide and grow. It induces mitoses, so that skin constantly produces and uses EGF, particularly when skin is damaged, such as in ultraviolet radiation and after surgery, and trauma for both healing and reduction of scar and keloid formation. When applied topically, EGF generates and replaces epithelial cells. EGF also promotes synthesis of proteins, accumulation of collagen and formation of blood vessels. Following sunlight injury and during the aging process, topical application of EGF replaces the existing low levels of dermal growth factors to achieve improvement in the quality of the skin, thereby reducing sagging skin and wrinkles. The antioxidants protect and repair damaged skin from free radicals while the growth factors to be used in combinations will promote epidermal cell renewal and thus ensue in repair of affected tissues, minimizing photodamage and mutations which promote cutaneous carcinogenesis.

Epidermal growth factor is a 53 amino acid polypeptide which stimulates messenger RNA, DNA and protein synthesis. In vitro it stimulates keratinocyte division and in vivo epidermal cell regeneration.

After cutaneous injury, residual epithelial cells proliferate in an organized fashion to regenerate an intact epidermis. Superficial wounds which do not result in total skin loss but retain at least a portion of the dermal layer, heal primarily by this process of epidermal regeneration. Epidermal growth factor induces replacement of cells by inducing mitosis. Many experiments, animal and human studies, have positively shown the beneficial effect of EGF in the process of wound repair. These clinical situations include partial thickness burns, skin graft donor sites, and chronic skin ulcers. It is also of use in healing radiation skin burns, surgical scars and in the repair process of cosmetic surgeries and cutaneous chemical peels.

Thiol rich yeast extracts also provide glutathione peroxidase and the sulphur groups to promote its synthesis and enhance the glutathione pathways. Thiol yeast extracts are used in concentrations ranging from 0.5% to 8%, most typically 3-5% and usually at 3.75 to 4.25% by weight.

As noted previously, the active ingredients described above can be incorporated in any suitable pharmacologically acceptable carrier which is suitable for topical administration to the human skin. As such, the pharmacologically acceptable carrier must be of sufficient purity and have sufficiently low toxicity to render it suitable for administration to a human noting that, typically, the carrier can represent up to 99.99% and typically from at least approximately 80% of the total composition.

Typical compositions for use herein include a wide variety of physical forms. These include, but are not limited to, solutions, lotions, creams, oils, gels, sticks, sprays, ointments, balms, patches and pastes. Generally, such carrier systems can be described as being solutions, creams, emulsions, gels, solids and aerosols.

Solvents are generally employed in the preparation of suitable topical compositions. Such solvents can either be aqueous or organic based and, in either case, the solvent must be capable of having dispersed or dissolved therein the above-described active components while not being irritating to the user. Water is a typical aqueous solvent while suitable organic solvents include propylene glycol, battalion glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, butanediol and mixtures thereof. Solvents can be included in the overall composition in amounts ranging from 0.1% to 99% and preferably from 2.0% to 75%. It is noted that compositions of the present invention can be produced in the form of an emollient. A wide variety of suitable emollients are known and may be used herein. In this regard, reference is made to U.S. Pat. No. 5,296,500, the disclosure of which is incorporated by reference.

Alternatively, the present composition can be formulated as a lotion containing from about 0.01% to 10% of the above described active ingredients. Further, the product can be formulated from a solution carrier system as a cream. A cream of the present invention would preferably comprise from about 0.1% to 15% and preferably from 1% to 5% of the above described active ingredients. Lotions and creams can be formulated as emulsions as well as solutions.

It is contemplated that as one embodiment, the active ingredients described above be used as a lotion or cream emulsion of the oil-in-water type or as a water-in-oil type, both of which being extremely well known in the cosmetic field. Multi-phase emulsions such as the water-in-oil type is disclosed in U.S. Pat. No. 4,254,105, the disclosure of which is incorporated herein by reference.

It is further contemplated that the active ingredients of the present invention be formulated from a solution carrier system as an ointment. An ointment may comprise a simple base of animal or vegetable oils or semi-solid hydrocarbons (oleaginous). Ointments may also comprise absorption ointment bases which absorb water to form emulsions. Ointment carriers may also be water soluble. An ointment may comprise from 1% to 99% of an emollient plus to about 0.1% to 99% of a thickening agent. Reference is again made to U.S. Pat. No. 5,296,500 and the citations contained therein for a more complete disclosure of the various ointment, cream and lotion formulations for use herein.

It is important to supply locally both glutathione and the synergistic antioxidants to restore epidermal glutathione levels and enhance the reparative antioxidant chain breaking reactions. It becomes imperative to prevent UV ray damage by prophylaxis with skin care (sun protection) products and appropriate clothing, plus the prevention of free radicals and their neutralization by locally applied chain-breaking antioxidant preparations, as proposed in the present application.

Claim 1 of 32 Claims

We claim:

1. A topical pharmaceutical composition for reducing the effects of ultraviolet radiation induced skin damage comprising as active ingredients an amount of reduced L-glutathione, selenium and epidermal growth factor to reduce ultraviolet radiation induced skin damage in a suitable carrier for topical application.

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